The present invention relates, inter alia, to novel variants (mutants) of parent Termamyl-like xcex1-amylases, notably variants exhibiting alterations in one or more properties (relative to the parent) which are advantageous with respect to applications of the variants in, in particular, industrial starch processing (e.g. starch liquefaction or saccharification).
xcex1-Amylases (xcex1-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides, and there is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes.
Among more recent disclosures relating to xcex1-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like xcex1-amylase which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens xcex1-amylase comprising the amino acid sequence shown in SEQ ID No. 4 herein and amino acids 301-483 of the C-terminal end of the B. licheniformis xcex1-amylase comprising the amino acid sequence shown in SEQ ID No. 2 herein (the latter being available commercially under the tradename Termamyl(trademark)), and which is thus closely related to the industrially important Bacillus xcex1-amylases (which in the present context are embraced within the meaning of the term xe2x80x9cTermamyl-like xcex1-amylasesxe2x80x9d, and which include, inter alia, the B. licheniformis, B. amyloliquefaciens and B. stearothermophilus xcex1-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like xcex1-amylase, variants of the parent Termamyl-like xcex1-amylase which exhibit altered properties relative to the parent.
As indicated above, the present invention relates, inter alia, to novel xcex1-amylolytic variants (mutants) of a Termamyl-like xcex1-amylase, in particular variants exhibiting altered properties which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like).
Alterations in properties which may be achieved in mutants of the invention are alterations in, e.g., substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile [such as increased stability at low (e.g. pH less than 6, in particular pH less than 5) or high (e.g. pH greater than 9) pH values], stability towards oxidation, Ca2+ dependency, specific activity, and other properties of interest. For instance, the alteration may result in a variant which, as compared to the parent Termamyl-like xcex1-amylase, has a reduced Ca2+ dependency and/or an altered pH/activity profile.
The invention further relates, inter alia, to DNA constructs encoding variants of the invention, to methods for preparing variants of the invention, and to the use of variants of the invention, alone or in combination with other xcex1-amylolytic enzymes, in various industrial processes, e.g. starch liquefaction.
The Termamyl-like xcex1-amylase
It is well known that a number of xcex1-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B. licheniformis xcex1-amylase comprising the amino acid sequence shown in SEQ ID No. 2 (commercially available as Termamyl(trademark)) has been found to be about 89% homologous with the B. amyloliquefaciens xcex1-amylase comprising the amino acid sequence shown in SEQ ID No. 4 and about 79% homologous with the B. stearothermophilus xcex1-amylase comprising the amino acid sequence shown in SEQ ID No. 6. Further homologous xcex1-amylases include an xcex1-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the xcex1-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31. Still further homologous xcex1-amylases include the xcex1-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the xcex1-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B. licheniformis xcex1-amylases are Optitherm(trademark) and Takatherm(trademark) (available from Solvay), Maxamyl(trademark) (available from Gist-brocades/Genencor), Spezym AA(trademark) (available from Genencor), and Keistase(trademark) (available from Daiwa).
Because of the substantial homology found between these xcex1-amylases, they are considered to belong to the same class of xcex1-amylases, namely the class of xe2x80x9cTermamyl-like xcex1-amylasesxe2x80x9d.
Accordingly, in the present context, the term xe2x80x9cTermamyl-like xcex1-amylasexe2x80x9d is intended to indicate an xcex1-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl(trademark), i.e. the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2 herein. In other words, a Termamyl-like xcex1-amylase is an xcex1-amylase which has the amino acid sequence shown in SEQ ID No. 2, No. 4 or No. 6 herein, or the amino acid sequence shown in SEQ ID No. 1 of WO 95/26397 (which amino acid sequence is shown in FIG. 1 and FIG. 2 herein) or in SEQ ID No. 2 of WO 95/26397 (which amino acid sequence is shown in FIG. 2 herein) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in FIG. 2 herein) or i) which displays at least 60%, such as at least 70%, e.g. at least 75%, or at least 80%, e.g. at least 85%, at least 90% or at least 95% homology with at least one of said amino acid sequences and/or ii) displays immunological cross-reactivity with an antibody raised against at least one of said xcex1-amylases, and/or iii) is encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified xcex1-amylases which are apparent from SEQ ID Nos. 1, 3 and 5 of the present application (which encoding sequences encode the amino acid sequences shown in SEQ ID Nos. 2, 4 and 6 herein, respectively), from SEQ ID No. 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in FIG. 1 herein and encodes the amino acid sequence shown in FIG. 1 herein) and from SEQ ID No. 5 of WO 95/26397, respectively.
In connection with property i), the xe2x80x9chomologyxe2x80x9d may be determined by use of any conventional algorithm, preferably by use of the GAP progamme from the GCG package version 7.3 (June 1993) using default values for GAP penalties [Genetic Computer Group (1991) Programme Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711].
Property ii) of the xcex1-amylase, i.e. the immunological cross reactivity, may be assayed using an antibody raised against, or reactive with, at least one epitope of the relevant Termamyl-like xcex1-amylase. The antibody, which may either be monoclonal or poly-clonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. The immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et al., 1989. In this respect, immunological cross-reactivity between the xcex1-amylases having the amino acid sequences SEQ ID Nos. 2, 4 and 6, respectively, has been found.
The oligonucleotide probe used in the characterization of the Termamyl-like xcex1-amylase in accordance with property iii) above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the xcex1-amylase in question. Suitable conditions for testing hybridization involve presoaking in 5xc3x97SSC and prehybridizing for 1 h at xcx9c40xc2x0 C. in a solution of 20% formamide, 5xc3x97Denhardt""s solution, 50 mM sodium phosphate, pH 6.8, and 50 xcexcg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 M ATP for 18 h at xcx9c40xc2x0 C., or other methods described by, e.g., Sambrook et al., 1989.
In the present context, xe2x80x9cderived fromxe2x80x9d is intended not only to indicate an xcex1-amylase produced or producible by a strain of the organism in question, but also an xcex1-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an xcex1-amylase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the xcex1-amylase in question. The term is also intended to indicate that the parent xcex1-amylase may be a variant of a naturally occurring xcex1-amylase, i.e. a variant which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring xcex1-amylase.
Parent Hybrid xcex1-amylases
The parent xcex1-amylase may be a hybrid xcex1-amylase, i.e. an xcex1-amylase which comprises a combination of partial amino acid sequences derived from at least two xcex1-amylases.
The parent hybrid xcex1-amylase may be one which on the basis of amino acid homology and/or immunological cross-reactivity and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like xcex1-amylase family. In this case, the hybrid xcex1-amylase is typically composed of at least one part of a Termamyl-like xcex1-amylase and part(s) of one or more other xcex1-amylases selected from Termamyl-like xcex1-amylases or non-Termamyl-like xcex1-amylases of microbial (bacterial or fungal) and/or mammalian origin.
Thus, the parent hybrid xcex1-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like xcex1-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial xcex1-amylase, or from at least one Termamyl-like and at least one fungal xcex1-amylase. The Termamyl-like xcex1-amylase from which a partial amino acid sequence derives may, e.g., be any of those specific Termamyl-like a xcex1-mylase referred to herein.
For instance, the parent xcex1-amylase may comprise a C-terminal part of an xcex1-amylase derived from a strain of B. licheniformis, and a N-terminal part of an xcex1-amylase derived from a strain of B. amyloliquefaciens or from a strain of B. stearothermophilus. For instance, the parent xcex1-amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis xcex1-amylase, and may, e.g. comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens xcex1-amylase having the amino acid sequence shown in SEQ ID No. 4 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2, or b) an amino acid segment corresponding to the 68 N-terminal amino acid residues of the B. stearothermophilus xcex1-amylase having the amino acid sequence shown in SEQ ID No. 6 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2.
The non-Termamyl-like xcex1-amylase may, e.g., be a fungal xcex1-amylase, a mammalian or a plant xcex1-amylase or a bacterial xcex1-amylase (different from a Termamyl-like xcex1-amylase). Specific examples of such xcex1-amylases include the Aspergillus oryzae TAKA xcex1-amylase, the A. niger acid xcex1-amylase, the Bacillus subtilis xcex1-amylase, the porcine pancreatic xcex1-amylase and a barley xcex1-amylase. All of these xcex1-amylases have elucidated structures which are markedly different from the structure of a typical Termamyl-like xcex1-amylase as referred to herein.
The fungal xcex1-amylases mentioned above, i.e. derived from A. niger and A. oryzae, are highly homologous on the amino acid level and generally considered to belong to the same family of xcex1-amylases. The fungal xcex1-amylase derived from Aspergillus oryzae is commercially available under the tradename Fungamyl(trademark).
Furthermore, when a particular variant of a Termamyl-like xcex1-amylase (variant of the invention) is referred toxe2x80x94in a conventional mannerxe2x80x94by reference to modification (e.g. deletion or substitution) of specific amino acid residues in the amino acid sequence of a specific Termamyl-like xcex1-amylase, it is to be understood that variants of another Termamyl-like xcex1-amylase modified in the equivalent position(s) (as determined from the best possible amino acid sequence alignment between the respective amino acid sequences) are encompassed thereby.
A preferred embodiment of a variant of the invention is one derived from a B. licheniformis xcex1-amylase (as parent Termamyl-like xcex1-amylase), e.g. one of those referred to above, such as the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2.
The construction of the variant of interest may be accomplished by cultivating a microorganism comprising a DNA sequence encoding the variant under conditions which are conducive for producing the variant. The variant may then subsequently be recovered from the resulting culture broth. This is described in detail further below.
The following discusses the relationship between mutations which may be present in variants of the invention, and desirable alterations in properties (relative to those a parent, Termamyl-like xcex1-amylase) which may result therefrom.
It is highly desirable to be able to decrease the Ca2+ dependency of a Termamyl-like xcex1-amylase. Accordingly, one aspect of the invention relates to a variant of a parent Termamyl-like xcex1-amylase, which variant exhibits xcex1-amylase activity and has a decreased Ca2+ dependency as compared to the parent xcex1-amylase. Decreased Ca2+ dependency will in general have the functional consequence that the variant exhibits a satisfactory amylolytic activity in the presence of a lower concentration of calcium ion in the extraneous medium than is necessary for the parent enzyme. It will further often have the consequence that the variant is less sensitive than the parent to calcium ion-depleting conditions such as those obtained in media containing calcium-complexing agents (such as certain detergent builders).
Decreased Ca2+ dependency of a variant of the invention may advantageously be achieved, for example, by increasing the Ca2+ binding affinity relative to that of the parent Termamyl-like xcex1-amylase; in other words the stronger the binding of Ca2+ in the enzyme, the lower the Ca2+ dependency.
It may be mentioned here that WO 96/23874 states that amino acid residues located within 10 xc3x85 from a sodium or calcium ion are believed to be involved in, or of importance for, the Ca2+ binding capability of the enzyme, and that in this connection the mutation N104D [of the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2, or an equivalent (N to D) mutation of an equivalent position in another Termamyl-like xcex1-amylase] is contemplated to be of particular interest with respect to decreasing the Ca2+ dependency of a Termamyl-like xcex1-amylase.
Other mutations mentioned in WO 96/23874 as being of possible importance in connection with Ca2+ dependency include mutations which are contemplated therein to achieve increased calcium binding (and/or thermostability of the enzyme) via stabilization of the C-domain (as defined in WO 96/23874) of the three-dimensional structure of a Termamyl-like xcex1-amylase via formation, for example, of cysteine bridges or salt bridges. Thus, WO 96/23874 discloses that the C-domain of the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2 may be stabilized by introduction of a cysteine bridge between domain A and domain C (as defined in WO 96/23874) by introduction of the following mutations:
A349C+I479C and/or L346C+I430C.
WO 96/23874 likewise discloses that a salt bridge may be obtained by introduction of one or more of the following mutations in the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2:
N457D,E
N457D,E+K385R
F350D,E+I430R,K
F350D,E+I411R,K
and that the calcium site of Domain C may be stabilized by replacing the amino acid residues H408 and/or G303 with any other amino acid residue, in particular by introducing one of the substitutions:
H408Q,E,N,D and/or G303N,D,Q,E
which are contemplated to provide better calcium binding or protection from calcium depletion.
(similar mutations in equivalent positions of other Termamyl-like xcex1-amylases being encompassed hereby).
Other substitution mutations (relative to B. licheniformis xcex1-amylase, SEQ ID No. 2) which are disclosed in WO 96/23874 as being of apparent importance, inter alia, in the context of reducing calcium dependency include the following: R23K, H156Y, A181T, A209V, R214, G310D and P345 (or equivalent mutations in equivalent positions in another Termamyl-like xcex1-amylase).
In the context of the present invention, further substitution mutations which appear to be of importance, inter alia, in relation to reduction of calcium dependency include the following mutations in Domain B (as defined in WO 96/23874):
A181E,D,Q,N,V (which appear to result in shielding of the outermost Ca2+ binding site in the junction region between Domain A and Domain B to some extent);
I201(bulkier amino acid); e.g. I201W,F,L (which appear to result in slight alterations in the geometry of the region in the immediate vicinity of the Ca2+xe2x80x94Na+xe2x80x94Ca2+ binding site(s) in the junction region between Domain A and Domain B, and in the geometry and/or size of a nearby hole/cavity); and
Y203E,Q (which are believed to result in stronger binding of the outermost Ca2+ ion in its binding site in the junction region between Domain A and Domain B); (or equivalent mutations in equivalent positions in another Termamyl-like xcex1-amylase).
WO 96/23874 discloses that it is contemplated to be possible to change the pH optimum of a Termamyl-like xcex1-amylase, or the enzymatic activity thereof at a given pH, by changing the pKa of the active site residues, and that this may be achieved, e.g., by changing the electrostatic interaction or hydrophobic interaction between functional groups of amino acid side chains of the amino acid residue to be modified and of its close surroundings.
In the context of the present invention, it is believed on the basis of electrostatic considerations [see, e.g., M. K. Gilson, Current Opinion in Structural Biology 5 (1995) pp. 216-223; and B. Honig and A. Nicholls, Science 268 (1995) pp. 1144-1149; and references given therein] and hygroscopicity considerations in relation to the three-dimensional structure of the Termamyl-like xcex1-amylase disclosed in WO 96/23874 that mutations of relevance, inter alia, for altering (increasing or decreasing) the pH optimum of a Termamyl-like xcex1-amylase include the following mutations or equivalents thereof [referring here to the sequence of B. licheniformis xcex1-amylase (SEQ ID NO 2)]:
Q9K,L,E; F11R,K,E; E12Q; D100N,L; V101H,R,K,D,E,F; V102A,T; I103H,K; N104R,K,D; H105R,K,D,E,W,F; L196R,K,D,E,F,Y; I212R,K,D,E; L230H,K,I; A232G,H,F,S,V; V233D; K234L,E; I236R,K,N,H,D,E; L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E; L270R,K,H,D,E; V283H,D; F284H; D285N,L; V286R,K,H,D,E; Y290R,E; V312R,K,D,E; F323H; D325N; N326K,H,D,L; H327Q,N,E,D,F; Q330L,E; G332D; Q333R,K,H,E,L; S334A,V,T,L,I,D; L335G,A,S,T,N; E336R+R375E; T337D,K; T338D,E; T339D; Q360K,R,E; D365N; G371D,R;
In the context of the present invention, mutations (amino acid substitutions) of importance with respect to achieving increased stability at low pH appear to include mutations corresponding to the following mutations in the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2:
mutations at positions H68, H91, H247, R305, K306, H382, K389, H405, H406, H450 or R483;
the mutations:
H140Y;
H142Y;
H156Y;
H159Y;
H140D+H142R;
H140K+H142D; or
H142Y+H156Y
as well as combinations of any two or more of these mutations.
A further aspect of the invention relates to a variant of a parent Termamyl-like xcex1-amylase, which variant is the result of one or more amino acid residues having been deleted from, substituted in or added to the parent xcex1-amylase so as to achieve increased thermostability of the variant.
In may be mentioned that in relation to achieving increased thermostability, WO 96/23874 discloses that a particularly interesting variant of a Termamyl-like xcex1-amylase comprises a mutation corresponding to one of the following mutations (using the numbering of the B. licheniformis xcex1-amylase amino acid sequence shown in SEQ ID NO 2):
L61W,V,F;
Y62W;
F67W;
K106R,F,W;
G145F,W
I212F,L,W,Y,R,K;
S151 replaced with any other amino acid residue and in particular with F,W,I or L;
R214W;
Y150R,K;
F143W; and/or
R146W.
WO 96/23874 further discloses in this connection that mutations corresponding to one or more of the following mutations in the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2 are of interest in relation to achieving increased thermostability relative to that of the parent xcex1-amylase:
L241I,F,Y,W; and/or
236L,F,Y,W
L7F,I,W
V259F,I,L
F284W
F350W
F343W
L427F,L,W
V481,F,I,L,W
In the context of the present invention, it can be seen from an alignment of the amino acid sequences of xcex1-amylases from various Bacillus species that B. licheniformis xcex1-amylase and B. amyloliquefaciens xcex1-amylase both contain an xe2x80x9cinsertionxe2x80x9d of three amino acids relative to, e.g., B. stearothermophilus xcex1-amylase.
From a model of the structure of B. licheniformis xcex1-amylase built on the basis of the three-dimensional structure of the Termamyl-like xcex1-amylase disclosed in WO 96/23784 (vide supra), taking into account the homology of B. licheniformis xcex1-amylase to the Termamyl-like xcex1-amylase in question, it can be seen that the above-mentioned xe2x80x9cinsertionxe2x80x9d lies within a part of the structure denoted xe2x80x9cloop 8xe2x80x9d in WO 96/23784, making this loop bulkier in B. licheniformis xcex1-amylase than in the Termamyl-like xcex1-amylase and resulting in a loop that protrudes from the structure, thereby possibly destabilizing the structure. It is therefore contemplated that deletion of one or more amino acids in the region in question in B. licheniformis or B. amyloliquefaciens xcex1-amylase will improve the thermostability of these xcex1-amylases.
Especially interesting in this connection is deletion of three amino acids within the partial sequence from T369 to I377 (referring to the amino acid sequence of B. licheniformis xcex1-amylase shown in SEQ ID No. 2), i.e. the partial sequence: T369-K370-G371-D372-S373-Q374-R375-E376-I377 (or the corresponding partial sequence in B. amyloliquefaciens xcex1-amylase). In addition to such deletions, substitution of one or more of the undeleted amino acids within the latter partial sequence may also be advantageous.
Preferable deletions of three amino acids in the partial sequence from T369 to I377 (in the B. licheniformis xcex1-amylase) are deletion of K370+G371+D372 (i.e. K370*+G371*+D372*) or deletion of D372+S373+Q374 (i.e. D372*+S373*+Q374*) (or equivalent deletions in the corresponding partial sequence in B. amyloliquefaciens xcex1-amylase).
Another type of mutation which would appear to be of value in improving the thermostability of these xcex1-amylases is substitution (replacement) of the entire partial amino acid sequence from T369 to I377 (referring to the sequence of the B. licheniformis xcex1-amylase) with one of the following partial sequences of six amino acids (sequence numbering increasing from left to right): I-P-T-H-S-V; I-P-T-H-G-V; and I-P-Q-Y-N-I (or one of the same substitutions of the corresponding partial sequence in B. amyloliquefaciens xcex1-amylase).
Other mutations which can apparently be of some importance in relation to achieving increased thermostability include amino acid substitutions at the following positions (referring to SEQ ID No. 2):
R169 (e.g. R169I,L,F,T);
R173 (especially R173I,L,F,T);
I201F;
I212F;
A209L,T; or
V208I
as well as combinations of any two or more of these mutations.
In the context of the invention, mutations which appear to be of particular relevance in relation to obtaining variants according to the invention having increased thermostability at acidic pH (pH less than 7) and/or at low Ca2+ concentration include mutations at the following positions (relative to B. licheniformis xcex1-amylase, SEQ ID No. 2):
H156, N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264, N265
It may be mentioned here that N and E amino acid residues, respectively, at positions corresponding to N109 and E211, respectively, in SEQ ID No. 2 constitute amino acid residues which are conserved in numerous Termamyl-like xcex1-amylases. Thus, for example, the corresponding positions of these residues in the amino acid sequences of a number of Termamyl-like xcex1-amylases which have already been mentioned (vide supra) are as follows:
Mutations of these conserved amino acid residues appear to be very important in relation to improving thermostability at acidic pH and/or at low calcium concentration, and the following mutations are of particular interest in this connection (with reference to the numbering of the B. licheniformis amino acid sequence shown in SEQ ID No. 2):
H156Y,D
N172R,H,K
A181T
N188P
N190L,F
H205C
D207Y
A209L,T,V
A210S
E211Q
Q264A E,L,K,S,T
N265A,S,T,Y
as well as any combination of two or more of these mutations.
An example of a particularly interesting double mutation in this connection is Q264S+N265Y.
In the starch liquefaction process it is desirable to use an xcex1-amylase which is capable of degrading the starch molecules into long, branched oligosaccharides, rather than an xcex1-amylase which gives rise to formation of shorter, branched oligosaccharides (like conventional Termamyl-like xcex1-amylases). Short, branched oligosaccharides (panose precursors) are not hydrolyzed satisfactorily by pullulanases, which are used after xcex1-amylase treatment in the liquefaction process, but before addition of a saccharifying amyloglucosidase (glucoamylase). Thus, in the presence of panose precursors, the product mixture present after the glucoamylase treatment contains a significant proportion of short, branched, so-called limit-dextrin, viz. the trisaccharide panose. The presence of panose lowers the saccharification yield significantly and is thus undesirable.
Thus, one aim of the present invention is to arrive at a mutant xcex1-amylase having appropriately modified starch-degradation characteristics but retaining the thermostability of the parent Termamyl-like xcex1-amylase.
It may be mentioned here that according to WO 96/23874, variants comprising at least one of the following mutations are expected to prevent cleavage close to the branching point:
V54L,I,F,Y,W,R,K,H,E,Q
D53L,I,F,Y,W
Y56W
Q333W
G57all possible amino acid residues
A52amino acid residues larger than A, e.g. A52W,Y,L,F,I.
In a further aspect of the present invention, important mutations with respect to obtaining variants exhibiting increased specific activity appear to include mutations corresponding to the following mutations in the B. licheniformis xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2:
mutations (amino acid substitutions) at positions S187 (especially S187D) or Q264 (e.g. Q264R,K,S);
mutations (substitutions) at position Y290 (especially Y290E,K);
the mutation V54I;
as well as combinations of any two or more of the latter mutations, or combinations of one, two or more of the latter mutations with the following multiple mutation:
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I
It may be preferred that a variant of the invention comprises one or more modifications in addition to those outlined above. Thus, it may be advantageous that one or more proline residues present in the part of the xcex1-amylase variant which is modified is/are replaced with a non-proline residue which may be any of the possible, naturally occurring non-proline residues, and which preferably is an alanine, glycine, serine, threonine, valine or leucine.
Analogously, it may be preferred that one or more cysteine residues present among the amino acid residues with which the parent xcex1-amylase is modified is/are replaced with a non-cysteine residue such as serine, alanine, threonine, glycine, valine or leucine.
Furthermore, a variant of the invention mayxe2x80x94either as the only modification or in combination with any of the above outlined modificationsxe2x80x94be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID No. 2 is replaced by an Asn and/or Gln, respectively. Also of interest is the replacement, in the Termamyl-like xcex1-amylase, of one or more of the Lys residues present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID No. 2 by an Arg.
It will be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.
Furthermore, it may be advantageous to introduce point-mutations in any of the variants described herein.
Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of xcex1-amylase-encoding DNA sequences, methods for generating mutations at specific sites within the xcex1-amylase-encoding sequence will be discussed.
The DNA sequence encoding a parent xcex1-amylase may be isolated from any cell or microorganism producing the xcex1-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the xcex1-amylase to be studied. Then, if the amino acid sequence of the xcex1-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify xcex1-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known xcex1-amylase gene could be used as a probe to identify xcex1-amylase-encoding clones, using hybridization and washing conditions of lower stringency.
Yet another method for identifying xcex1-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming xcex1-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for xcex1-amylase, thereby allowing clones expressing the xcex1-amylase to be identified.
Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by S. L. Beaucage and M. H. Caruthers (1981) or the method described by Matthes et al. (1984). In the phosphoroamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al. (1988).
Once an xcex1-amylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the xcex1-amylase-encoding sequence, is created in a vector carrying the xcex1-amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.
Another method for introducing mutations into xcex1-amylase-encoding DNA sequences is described in Nelson and Long (1989). It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.
Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence in question, or within the whole gene.
WO 96/23874 discloses that in connection with achieving improved binding of a substrate (i.e. improved binding of a carbohydrate species, such as amylose or amylopectin) by a Termamyl-like xcex1-amylase variant, modified (e.g. higher) substrate specificity and/or modified (e.g. higher); specificity with respect to cleavage (hydrolysis) of substrate, the following codon positions for the amino acid sequence shown in SEQ ID NO 2 (or equivalent codon positions for another parent Termamyl-like xcex1-amylase in the context of the invention) appear to be particularly appropriate for targetting:
13-18
50-56
70-76
102-109
163-172
189-199
229-235
360-364
327-335
For an xcex1-amylase to be used in a starch liquefaction process it is of particular interest that it be thermostable and able to function at low pH and low calcium concentrations. In order to improve these properties of a parent Termamyl-like xcex1-amylase, in particular the B. licheniformis xcex1-amylase or a variant or hybrid thereof, random mutagenesis (preferably by use of doped or spiked oligonucleotide primers) followed by appropriate selection of the resulting mutated enzymes may be performed. The direction of selection of regions to randomize and selection of doping are based primarily on stabilization of calcium ions already present, and on improvement in residue/residue or domain/domain electrostatic interactions at low pH. In addition, the regions which have been shown to include positions important for achieving good starch liquefaction performance may be selected.
In order to prepare a variant of a parent Termamyl-like xcex1-amylase having the above properties, at least one of the following regions may advantageously be subjected to random mutagenesis (the numbering of the amino acid residues being as in SEQ ID No. 2):
Preferably, two, three or four of the above regions are subjected to random mutagenesis in the construction of a novel xcex1-amylase variant of the invention. For instance, the following combinations of regions are suitably subjected to random mutagenesis:
VIII+IX
VIII+IX+II
II+III+IV
IV+I
Furthermore, it is preferred that the mutagenesis is carried out by use of doped or spiked oligonucleotides. The doping is preferably done so as to introduce amino acids contributing to improved stability at low pH and reduced calcium dependency at low pH of the resulting xcex1-amylase variant. Furthermore, when selecting the doping scheme, the possibility of introducing Asn and Gln residues should generally be avoided, since Asn and Gln residues in general are associated with instability at low pH. Preferably, when a Pro residue can be inserted with potential benefits (e.g. as assessed from protein-structural considerations), the doping scheme is prepared to include a preference for introduction of a Pro residue.
The parent Termamyl-like xcex1-amylase to be subjected to random mutagenesis according to the above principle may be any wild type xcex1-amylase or a variant thereof containing one or more mutations. The parent may be a hybrid between at least two xcex1-amylases as explained in further detail herein. Preferably, the parent xcex1-amylase is a mutant of the B. licheniformis xcex1-amylase having the sequence shown in SEQ ID No. 2 containing at least one mutation, and preferably multiple mutations. The parent xcex1-amylase may alternatively be a hybrid xcex1-amylase which contains at least a part of the B. licheniformis (SEQ ID No. 2) xcex1-amylase. Specific examples of parent xcex1-amylases suited to mutagenesis according to the above-described principles include: variants of the B. licheniformis (SEQ ID No. 2) xcex1-amylase which contain at least one of, i.e. one, two, three, four or all five of, the mutations H156Y, A181T, N190F, A209V and Q264S; hybrid xcex1-amylases which contain a part of the B. licheniformis (SEQ ID No. 2) xcex1-amylase, preferably a C-terminal part thereof, such as amino acids 35-483 thereof, and a part of another Termamyl-like xcex1-amylase such as B. amyloliquefaciens (SEQ ID No. 4) xcex1-amylase, preferably an N-terminal part thereof such as the first 38 amino acid residues thereof.
In relation to the above, a further aspect of the present invention relates to a method for generating a variant of a parent Termamyl-like xcex1-amylase, which variant exhibits increased stability at low pH and at low calcium concentration relative to the parent, the method comprising:
(a) subjecting a DNA sequence encoding the parent Termamyl-like xcex1-amylase to random mutagenesis,
(b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and
(c) screening for host cells expressing a mutated xcex1-amylase which has increased stability at low pH and low calcium concentration relative to the parent xcex1-amylase.
Step (a) of the latter method of the invention is preferably performed using doped primers, as described in the working examples herein (vide infra).
The random mutagenesis of a DNA sequence encoding a parent xcex1-amylase to be performed in accordance with step a) of the above-described method of the invention may conveniently be performed by use of any method known in the art.
For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents.
The mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties.
When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the amylolytic enzyme by any published technique, using e.g. PCR, LCR or any DNA polymerase and ligase.
Preferably, the doping is carried out using xe2x80x9cconstant random dopingxe2x80x9d, in which the percentage of wild-type and mutation in each position is predefined. Furthermore, the doping may be directed to have a preference for the introduction of certain nucleotides, and thereby a preference for introduction of one or more specific amino acid residues. The doping may, e.g., be made so as to allow for the introduction of 90% wild type and 10% mutations in each position. An additional consideration in choice of doping scheme is genetic as well as protein-structural constraints. The doping scheme may be made by using the DOPE program (see the working examples herein) which, inter alia, ensures that introduction of stop codons is avoided.
When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent xcex1-amylase enzyme is subjected to PCR under conditions that increase the mis-incorporation of nucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp. 11-15).
A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133, 1974, pp. 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the amylolytic enzyme by e.g. transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may subsequently be transformed into the expression organism.
The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent amylolytic enzyme. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenizing agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
In some cases it may be convenient to amplify the mutated DNA sequence prior to the expression step (b) or the screening step (c) being performed. Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligdnucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.
Subsequent to the incubation with or exposure to the mutagenizing agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated
DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are the following: grampositive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gramnegative bacteria such as E.coli. 
The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.
Localized Random Mutagenesis
The random mutagenesis may advantageously be localized to a part of the parent xcex1-amylase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
The localized random mutagenesis is conveniently performed by use of PCR-generated mutagenesis techniques as described above, or any other suitable technique known in the art.
Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g. by being inserted into a suitable vector, and said part may subsequently be subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
With respect to the screening step in the above-mentioned method of the invention, this may conveniently performed by use of an assay as described in connection with Example 2 herein.
With regard to screening in general, a filter assay based on the following is generally applicable:
A microorganism capable of expressing the mutated amylolytic enzyme of interest is incubated on a suitable medium and under suitable conditions for the enzyme to be secreted, the medium being provided with a double filter comprising a first protein-binding filter and on top of that a second filter exhibiting a low protein binding capability. The microorganism is located on the second filter. Subsequent to the incubation, the first filter comprising enzymes secreted from the microorganisms is separated from the second filter comprising the microorganisms. The first filter is subjected to screening for the desired enzymatic activity and the corresponding microbial colonies present on the second filter are identified.
The filter used for binding the enzymatic activity may be any protein binding filter e.g. nylon or nitrocellulose. The top-filter carrying the colonies of the expression organism may be any filter that has no or low affinity for binding proteins e.g. cellulose acetate or Durapore(trademark). The filter may be pretreated with any of the conditions to be used for screening or may be treated during the detection of enzymatic activity.
The enzymatic activity may be detected by a dye, fluorescence, precipitation, pH indicator, IR-absorbance or any other known technique for detection of enzymatic activity.
The detecting compound may be immobilized by any immobilizing agent e.g. agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents.
xcex1-Amylase activity is detected by Cibacron Red labelled amylopectin, which is immobilized on agarose. For screening for variants with increased thermal and high-pH stability, the filter with bound xcex1-amylase variants is incubated in a buffer at pH 10.5 and 60xc2x0 or 65xc2x0 C. for a specified time, rinsed briefly in deionized water and placed on the amylopectin-agarose matrix for activity detection. Residual activity is seen as lysis of Cibacron Red by amylopectin degradation. The conditions are chosen to be such that activity due to the xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2 can barely be detected. Stabilized variants show, under the same conditions, increased colour intensity due to increased liberation of Cibacron Red.
For screening for variants with an activity optimum at a lower temperature and/or over a broader temperature range, the filter with bound variants is placed directly on the amylopectin-Cibacron Red substrate plate and incubated at the desired temperature (e.g. 4xc2x0 C., 10xc2x0 C. or 30xc2x0 C.) for a specified time. After this time activity due to the xcex1-amylase having the amino acid sequence shown in SEQ ID No. 2 can barely be detected, whereas variants with optimum activity at a lower temperature will show increase amylopectin lysis. Prior to incubation onto the amylopectin matrix, incubation in all kinds of desired mediaxe2x80x94e.g. solutions containing Ca2+, detergents, EDTA or other relevant additivesxe2x80x94can be carried out in order to screen for changed dependency or for reaction of the variants in question with such additives.
The testing of variants of the invention may suitably be performed by determining the starch-degrading activity of the variant, for instance by growing host cells transformed with a DNA sequence encoding a variant on a starch-containing agarose plate and identifying starch-degrading host cells. Further testing as to altered properties (including specific activity, substrate specificity, cleavage pattern, thermoactivation, pH optimum, pH dependency, temperature optimum, and any other para-meter) may be performed in accordance with methods known in the art.
According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
The recombinant expression vector carrying the DNA sequence encoding an xcex1-amylase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding an xcex1-amylase variant of the invention, especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis xcex1-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens xcex1-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral xcex1-amylase, A. niger acid stable xcex1-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the xcex1-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.
While intracellular expression may be advantageous in some respects, e.g. when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus xcex1-amylases mentioned herein comprise a pre-region permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.
The procedures used to ligate the DNA construct of the invention encoding an xcex1-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al. (1989)).
The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an xcex1-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.
Examples of suitable bacteria are grampositive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.
The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
In a yet further aspect, the present invention relates to a method of producing an xcex1-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.
The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the xcex1-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
The xcex1-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
The xcex1-amylase variants of this invention possesses valuable properties allowing for a variety of industrial applications. In particular, enzyme variants of the invention are applicable as a component in washing, dishwashing and hard-surface cleaning detergent compositions. Numerous variants are particularly useful in the production of sweeteners and ethanol from starch, and/or for textile desizing. Conditions for conventional starch-conversion processes, including starch liquefaction and/or saccharification processes, are described in, e.g., U.S. Pat. No. 3,912,590 and in EP patent publications Nos. 252,730 and 63,909.
Production of Sweeteners from Starch
A xe2x80x9ctraditionalxe2x80x9d process for conversion of starch to fructose syrups normally consists of three consecutive enzymatic processes, viz. a liquefaction process followed by a saccharification process and an isomerization process. During the liquefaction process, starch is degraded to dextrins by an xcex1-amylase (e.g. Termamyl(trademark)) at pH values between 5.5 and 6.2 and at temperatures of 95-160xc2x0 C. for a period of approx. 2 h. In order to ensure an optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions).
After the liquefaction process the dextrins are converted into dextrose by addition of a glucoamylase (e.g. AMG(trademark)) and a debranching enzyme, such as an isoamylase or a pullulanase (e.g. Promozyme(trademark)). Before this step the pH is reduced to a value below 4.5, maintaining the high temperature (above 95xc2x0 C.), and the liquefying xcex1-amylase activity is denatured. The temperature is lowered to 60xc2x0 C., and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.
After the saccharification process the pH is increased to a value in he range of 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immmobilized glucoseisomerase (such as Sweetzyme(trademark))
At least 3 enzymatic improvements of this process could be envisaged. All three improvements could be seen as individual benefits, but any combination (e.g. 1+2, 1+3, 2+3 or 1+2+3) could be employed:
Improvement 1
Reduction of the calcium dependency of the liquefying alpha-amylase.
Addition of free calcium is required to ensure adequately high stability of the xcex1-amylase, but free calcium strongly inhibits the activity of the glucoseisomerase and needs to be removed, by means of an expensive unit operation, to an extent which reduces the level of free calcium to below 3-5 ppm. Cost savings could be obtained if such an operation could be avoided and the liquefaction process could be performed without addition of free calcium ions.
To achieve that, a less calcium-dependent Termamyl-like xcex1-amylase which is stable and highly active at low concentrations of free calcium ( less than 40 ppm) is required. Such a Termamyl-like xcex1-amylase should have a pH optimum at a pH in the range of 4.5-6.5, preferably in the range of 4.5-5.5.
Improvement 2
Reduction of formation of unwanted Maillard products.
The extent of formation of unwanted Maillard products during the liquefaction process is dependent on the pH. Low pH favours reduced formation of Maillard products. It would thus be desirable to be able to lower the process pH from around pH 6.0 to a value around pH 4.5; unfortunately, all commonly known, thermostable Termamyl-like xcex1-amylases are not very stable at low pH (i.e. pH  less than 6.0) and their specific activity is generally low.
Achievement of the above-mentioned goal requires a Termamyl-like xcex1-amylase which is stable at low pH in the range of 4.5-5.5 and at free calcium concentrations in the range of 0-40 ppm, and which maintains a high specific activity.
Improvement 3
It has been reported previously (U.S. Pat. No. 5,234,823) that when saccharifying with A. niger glucoamylase and B. acidopullulyticus pullulanase, the presence of residual xcex1-amylase activity from the liquefaction process can lead to lower yields of dextrose if the xcex1-amylase is not inactivated before the saccharification stage. This inactivation can typically be carried out by adjusting the pH to below 4.3 at 95xc2x0 C., before lowering the temperature to 60xc2x0 C. for saccharification.
The reason for this negative effect on dextrose yield is not fully understood, but it is assumed that the liquefying xcex1-amylase (for example Termamyl(trademark) 120 L from B. licheniformis) generates xe2x80x9climit dextrinsxe2x80x9d (which are poor substrates for B. acidopullulyticus pullulanase) by hydrolysing 1,4-xcex1-glucosidic linkages close to and on both sides of the branching points in amylopectin. Hydrolysis of these limit dextrins by glucoamylase leads to a build-up of the trisaccharide panose, which is only slowly hydrolysed by glucoamylase.
The development of a thermostable xcex1-amylase which does not suffer from this disadvantage would be a significant process improvement, as no separate inactivation step would be required.
If a Termamyl-like, low-pH-stable xcex1-amylase is developed, an alteration of the specificity could be an advantage needed in combination with increased stability at low pH.
The methodology and principles of the present invention make it possible to design and produce variants according to the invention having required properties as outlined above. In this connection, particularly interesting mutations are mutations in a Termamyl-like xcex1-amylase [for example Termamyl(trademark) itself (B. licheniformis xcex1-amylase; SEQ ID No. 2); or a Termamyl-like xcex1-amylase having an N-terminal amino acid sequence (i.e. the partial sequence up to the amino acid position corresponding to position 35 in Termamyl(trademark)) which is identical to that in B. amyloliquefaciens xcex1-amylase (SEQ ID No. 4), i.e. a Termamyl-like xcex1-amylase having the following N-terminal sequence relative to amino acid sequence of Termamyl(trademark): A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I, where an asterisk (*) indicates deletion of the amino acid residue in question] at positions corresponding to any of the following positions in Termamyl(trademark):
H133
H156
A181
A209
G310
H450
V128
N104
V54
S187
H293
A294
(where each of the latter amino acid residues may be replaced by any other amino acid residue, i.e. any other residue chosen among A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V), as well as the following triple deletions:
K370*+G371*+D372*
D372*+S373*+Q374*
Particularly preferred substitutions at the above-indicated positions are the following:
H133I
H156Y
A181T
A209V
G310D
H450Y
V128E
N104D
V54W,Y,F,I,L
S187D
H293Y
A294V.
Any combination of one or more (i.e. one, two, three, four. . . etc.) of the above indicated mutations may appropriately be effected in a Termamyl-like xcex1-amylase in the context in question, and particularly interesting variants of the invention in the context of achieving one or more of the above-mentioned improvements in relation to the starch liquefaction behaviour of xcex1-amylases include variants comprising combinations of multiple mutations corresponding to the following combinations of mutations in Termamyl(trademark) (SEQ ID No. 2) itself:
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54W+S187D+H293Y+A294V+K370*+G371*+D372*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54W+S187D+H293Y+A294V+D372*+S373*+Q374*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54Y+S187D+H293Y+A294V+K370*+G371*+D372*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54Y+S187D+H293Y+A294V+D372*+S373*+Q374*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54F+S187D+H293Y+A294V+K370*+G371*+D372*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54F+S187D+H293Y+A294V+D372*+S373*+Q374*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54I+S187D+H293Y+A294V+K370*+G371*+D372*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54I+S187D+H293Y+A294V+D372*+S373*+Q374*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54L+S187D+H293Y+A294V+K370*+G371*+D372*;
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54L+S187D+H293Y+A294V+D372*+S373*+Q374*;
Further interesting variants of the invention in this context include variants comprising single or multiple mutations corresponding to the following single or multiple mutations in Termamyl(trademark) itself:
mutations (amino acid substitutions) at positions N172 (e.g. N172R,K), S187 (e.g. S187D), N188 (e.g. N188P), N190 (e.g. N190L,F), H205 (e.g. H205C), D207 (e.g. D207Y), A210 (e.g. A210S), Q264 (e.g. Q264S) or N265 (e.g. N265Y);
the following multiple mutations;
H156Y+A181T+A209V;
H156Y+A181T+N190F+A209V+Q264S
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A209V;
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N190F+A209V; or
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N190F+A209V+Q264S
as well as combinations of any two or more of the latter single or multiple mutations.
As already indicated, numerous variants according to the invention a re particularly well suited for use in starch conversion, e.g. in starch liquefaction. In this connection, a further aspect of the present invention relates to compositions comprising a mixture of:
(i) the xcex1-amylase from B. licheniformis having the sequence shown in SEQ ID No. 2 with one or more variants (mutant xcex1-amylases) according to the invention derived from (as the parent Termamyl-like xcex1-amylase) the B. stearothermophilus xcex1-amylase having the sequence shown in SEQ ID No. 6; or
(ii) the xcex1-amylase from B. stearothermophilus having the sequence shown in SEQ ID No. 6 with one or more variants (mutant xcex1-amylases) according to the invention derived from one or more other parent Termamyl-like xcex1-amylases (e.g. from the B. licheniformis xcex1-amylase having the sequence shown in SEQ ID No. 2, or from one of the other parent Termamyl-like xcex1-amylases specifically referred to herein); or
(iii) one or more variants (mutant xcex1-amylases) according to the invention derived from (as the parent Termamyl-like xcex1-amylase) the B. stearothermophilus xcex1-amylase having the sequence shown in SEQ ID No. 6 with one or more variants (mutant xcex1-amylases) according to the invention derived from one or more other parent Termamyl-like xcex1-amylases (e.g. from the B. licheniformis xcex1-amylase having the sequence shown in SEQ ID No. 2, or from one of the other parent Termamyl-like xcex1-amylases specifically referred to herein).
Preferred mutations in a variant of B. stearothermophilus xcex1-amylase to be incorporated in such a mixture include substitutions at N193 and/or at E210, and/or the pairwise deletions R179*+G180* or I181*+G182* (using the numbering of the amino acid sequence for this particular xcex1-amylase).
Compositions of one of the latter types, containing B. stearothermophilus xcex1-amylase or a variant thereof according to the invention, appear to have great potential for use in starch liquefaction. The ratio (expressed, e.g., in terms of mg of active amylolytic protein per liter of liquid medium) between the individual xcex1-amylolytic components of a given mixture will depend on the exact nature and properties of each component.
As mentioned above, variants of the invention may suitably be incorporated in detergent compositions. Reference is made, for example, to WO 96/23874 and WO 97/07202 for further details concerning relevant ingredients of detergent compositions (such as laundry or dishwashing detergents), appropriate methods of formulating the variants in such detergent compositions, and for examples of relevant types of detergent compositions.
Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another xcex1-amylase.
xcex1-Amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-1 mg (calculated as pure, active enzyme protein) of xcex1-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.